1. Field of the Invention
This invention relates to the field of radiolabeled proteins for diagnostic and therapeutic applications and, more particularly, to chelating agents useful for coupling metal ions to biologically active molecules.
2. Background of the Invention
The attachment of radiometals to proteins, especially antibodies and antibody fragments results in the formation of new radiodiagnostic and radiotherapeutic agents. The use of pure, homogeneous monoclonal antibodies for this purpose has been recognized as an important advancement in nuclear medicine.
The performance of the radiometal-protein conjugates depends on a number of factors, including the stability of the metal chelate complex in regard to loss of the radiometal from the chelate in vivo and the ability of the conjugates to localize to the target tissue. As it is desired that the radioactive agent not accumulate in any but the target tissues, the performance of such agents also depends on the extent and rate at which the agents are eventually cleared from non-target tissue.
Monoclonal antibodies are highly specific and can be used as vehicles to deliver substances to specific target sites. Numerous antibodies have been developed with affinity for targets such as myosin, fibrinogen, atherosclerotic tissue and tumors, to name just a few, and work in this area continues.
Radiometals may be attached to proteins and antibodies through the use of bifunctional chelating agents. The bifunctional agent is selected such that it is capable of binding radiometals by chelation as well as forming a linkage to the protein. For example, diethylenetriamine pentaacetic acid (DTPA) has been conjugated to an antimyosin antibody, and the proteinbound DTPA used to chelate In-111 (Khaw, et al., Science, 209, 295 (1980), See also Krejcarek, et al., Biochem. Biophys. Res. Comm., 77, 581 (1977) and Childs, R. L. and Hnatowich, D. J., J. Nuc. Med., 26, 293 (1985)).
The stability of the metal-chelator complex is important, as leakage of the radiometal from the protein-chelate conjugate can result in a high background during imaging procedures and damage to non-targeted tissues in radioimmunotherapy. U.S. Pat. No. 4,472,509 discloses bifunctional DTPA derivatives which form metal complexes with increased in vivo stability. The ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, forms remarkably stable complexes with a number of metals, (Stetter, H. and Frank, W., Angew. Chem. Int Ed. Engl., 15, 686, (1976), Desreux, J. F., Loncin, M. F., and Merciny, E., Inorg. Chem. 25, 2646 (1986)). The 2-(p-nitrobenzyl) derivative of DOTA has been described (Meares, C. F., Moi, M. K. and De Nardo, S.J., J. Am. Chem. Soc. 110, 6266 (1988)) and the yttrium complex was determined to be inert to loss of the metal from the complex to serum proteins. A series of 1-substituted analogs of DOTA have been described (Tweedle, M. F. et. al., Eur. Pat. Appl. EP 232, 751). The gadolinium complex of one such analog, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, was reported to be inert to reaction with endogenously available ions (Tweedle, M. F. et al., J. Nuc. Med., 28, 705 (1988)).
Bifunctional chelators have been coupled to proteins and antibodies at a number of chemical sites. Lysyl side chain amino groups have been modified (EPO Publication No. 188, 256) and
chelators have been site-selectively attached to oxidized antibody carbohydrate moieties (U.S. Pat. No. 4,671,958). Chelators have also been attached by reaction with free sulfhydryl groups (U.S. Pat. No. 4,659,839, U.S. Pat. No. 4,671,958, EPO Publication No. 173, 629 and U.S. application Ser. No. 199,931). Site-selective antibody modification is advantageous as the chelator may be placed at points removed from the antigen-binding region thus preserving the immunoreactivity of the protein.
The accumulation of radiolabeled antibodies in non-target tissue is a factor which limits the dosage at which the conjugates may be used. The clearance of radio metal labeled antibodies by the use of a cleavable bifunctional chelating agent has been addressed. DTPA derivatives containing cleavable functionalities have been used to form conjugates between indium-111 and antibodies (Park, C. H. et al., J. Nuc. Med., 27, 959 (1986), 28, 572 (1987), 29, 889 (1988), Meares, C. F., et al., Eur. J. Nuc. Med., 12, 455 (1986), Meares., C. F., et al., Int. J. Cancer Supp. 2, 99 (1988)). In general, these compositions showed an increased clearance rate in mice.
A need for bifunctional chelating agents which are capable of forming stable metal complexes, and optionally which contain a cleavable linking unit, continues to exist in the field of nuclear medicine.